Soy lecithin supplementation alters macrophage phagocytosis and lymphocyte response to concanavalin A: a study in alloxan-induced diabetic rats

Dietary soy lecithin supplementation decreases hyperlipidemia and influences lipid metabolism. Although this product is used by diabetic patients, there are no data about the effect of soy lecithin supplementation on the immune system. The addition of phosphatidylcholine, the main component of lecithin, to a culture of lymphocytes has been reported to alter their function. If phosphatidylcholine changes lymphocyte functions in vitro as previously shown, then it could also affect immune cells in vivo. In the present study, the effect of dietary soy lecithin oil macrophage phagocytic capacity and on lymphocyte number in response to concanavalin A (ConA) stimulation was investigated in non-diabetic and alloxan-induced diabetic rats. Supplementation was carried Out daily with 2 g kg(-1) b.w. lecithin during 7 days. After that, blood was drawn from fasting rats and peritoneal macrophages and mesenteric lymph node lymphocytes were collected to determine the phospholipid content. Plasma triacylglycerol (TAG), total and HDL cholesterol and glucose levels were also determined. Lymphocytes were stimulated by Conk The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye reduction method and flow cytometry were employed to evaluate lymphocyte metabolism and cell number, respectively. Soy lecithin supplementation significantly increased both macrophage phagocytic capacity (+29%) in non-diabetic rats and the lymphocyte number in diabetic rats (+92%). It is unlikely that plasma lipid levels indirectly affect immune cells, since plasma cholesterol, TAG, or phospholipid content was not modified by lecithin supplementation. In Conclusion, lymphocyte and macrophage function were altered by lecithin supplementation, indicating ail immunomodulatory effect of phosphatidylcholine. Copyright (C) 2008 John Wiley & Sons, Ltd.

Glucose metabolism by lymphocytes, macrophages, and tumor cells from Walker 256 tumor-bearing rats supplemented with fish oil for one generation

Here we investigated the effect of lifelong supplementation of the diet with coconut fat (CO, rich in saturated fatty acids) or fish oil (170, rich in n-3 polyunsaturated fatty acids) on tumor growth and lactate production from glucose in Walker 256 tumor cells, peritoneal macrophages, spleen, and gut-associated lymphocytes. Female Wistar rats were supplemented with CO or FO prior to mating and then throughout pregnancy and gestation and then the male offspring were supplemented from weaning until 90 days of age. Then they were inoculated subcutaneously with Walker 256 tumor cells. Tumor weight at 14 days in control rats (those fed standard chow) and CO supplemented was approximately 30 g. Supplementation of the diet with FO significantly reduced tumor growth by 76%. Lactate production (nmol h(-1) mg(-1) protein) from glucose by Walker 256 cells in the group fed regular chow (W) was 381.8 +/- 14.9. Supplementation with coconut fat (WCO) caused a significant reduction in lactate production by 1.6-fold and with fish oil (WFO) by 3.8-fold. Spleen lymphocytes obtained from W and WCO groups had markedly increased lactate production (553 +/- 70 and 635 +/- 150) when compared to non-tumor-bearing rats (similar to 260 +/- 30). FO supplementation reduced significantly the lactate production (297 +/- 50). Gut-associated lymphocytes obtained from W and WCO groups increased lactate production markedly (280 +/- 31 and 276 +/- 25) when compared to non-tumor-bearing rats (similar to 90 +/- 18). FO supplementation reduced significantly the lactate production (168 +/- 14). Lactate production by peritoneal macrophages was increased by tumor burden but there was no difference between the groups fed the various diets. Lifelong consumption of FO protects against tumor growth and modifies glucose metabolism in Walker tumor cells and lymphocytes but not in macrophages. Copyright (C) 2008 John Wiley & Sons, Ltd.

Liver glycogen metabolism during short-term insulin-induced hypoglycemia in fed rats

The activities of glycogen phosphorylase and synthase during infusions of glucagon, isoproterenol, or cyanide in isolated liver of fed rats submitted to short-term insulin-induced hypoglycemia (IIH) was investigated. A condition of hyperinsulinemia/hypoglycemia was obtained with an intraperitoneal injection of regular insulin (1.0 U kg(-1)). The control group received ip saline. The experiments were carried out 60 min after insulin (IIH group) or saline (COG group) injection. The rats were anesthetized and after laparotomy, blood was collected from the vena cava for glucose and insulin measurements. The liver was their infused with glucagon (1 nM), isoproterenot (2 mu M), or cyanide (0.5 mM) during 20 min and a sample of the organ was collected for determination of the activities of glycogen phosphorylase and synthase 5 min after starting and 10 min after stopping the infusions. The infusions of cyanide, glucagons, and isoproterenol did not change the activities of glycogen synthase and glycogen phosphorylase. However, glycogen catabolism was decreased during the infusions of glucagon and isoproterenol in IIH rats, being more intense with isoproterenol (p < 0.05), than glucagon. It was concluded that short-term IIH promoted changes in the liver responsiveness of glycogen degradation induced by glucagon and isoproterenol without a change in the activities of glycogen phosphorylase and synthase. Copyright (c) 2008 John Wiley & Sons, Ltd.

Effect of oleic and linoleic acids on the inflammatory phase of wound healing in rats

Inflammation is a crucial step for the wound healing process. The effect of linoleic and oleic acids on the inflammatory response of the skin during the healing process and on the release of pro-inflammatory cytokines by rat neutrophils in vitro was investigated. A wound in the dorsal surface of adult rats was performed and fatty acids were then topically administered. Both oleic and linoleic acids increased the wound healing tissue mass. The total protein and DNA contents of the wounds were increased by the treatment with linoleic acid. The treatments with oleic and linoleic acids did not affect vascular permeability. However, the number of neutrophils in the wounded area and air pouches was increased and the thickness of the necrotic cell layer edge around the wound was decreased. A dose-dependent increase in vascular endothelial growth factor-alpha (VEGF-alpha) and interleukin-1 beta (IL-1 beta) by neutrophils incubated in the presence of oleic and linoleic acid was observed. Oleic acid was able to stimulate also the production of cytokine-induced neutrophil chemoattractant in inflammation 2 alphalbeta (CINC-2 alpha/beta). This pro-inflammatory effect of oleic and linoleic acids may speed up the wound healing process. Copyright (c) 2007 John Wiley & Sons, Ltd.

Participation of the liver gluconeogenesis in the glibenclamide-induced hypoglycaemia in rats

We previously demonstrated an increased liver gluconeogenesis (LG) during insulin-induced hypoglycaemia. Thus, an expected effect of sulphonylureas induced hypoglycaemia (SIH) could be the activation of LG. However, sulphonylureas infused directly in to the liver inhibits LG. Considering these opposite effects we investigated herein LG in rats submitted to SIH. For this purpose, 24 h fasted rats that received glibenclamide (10 mg kg(-1)) were used (SIH group). Control group received oral saline. Glycaemia at 30, 60, 90, 120 and 150 min after oral administration of glibenclamide were evaluated. Since the lowest glycaemia was obtained 120 min after glibenclamide administration, this time was chosen to investigate LG in situ perfused livers. The gluconeogenesis from precursors that enters in this metabolic pathway before the mitochondrial step, i.e. L-alanine (5 mM), L-lactate (2 mM), pyruvate (5 mM) and L-glutamine were decreased (p < 0.05). However, the gluconeogenic activity using glycerol (2 mM), which enters in the gluconeogenesis after the mitochondrial step was maintained. Taken together, the results suggest that the inhibition of LG promoted by SIH overcome the activation of this metabolic pathway promoted by IIH and could be attributed, at least in part, to its effect on mitochondrial function. Copyright (C) 2011 John Wiley & Sons, Ltd.

Testosterone suppresses oxidative stress in human neutrophils

The in vitro effect of testosterone on human neutrophil function was investigated. Blood neutrophils from healthy male subjects were isolated and treated with 10 nM, 0.1 and 10 mu M testosterone for 24 h. As compared with untreated cells, the testosterone treatment produced a significant decrease of superoxide production as indicated by the measurement of extra- and intracellular superoxide content. An increment in the production of nitric oxide was observed at 0.1 and 10 mu M testosterone concentrations, whereas no effect was found for 10 nM. Intracellular calcium mobilization was significantly increased at 10 nM, whereas it was reduced at 10 mu M testosterone. There was an increase in phagocytic capacity at 10 nM and a decrease of microbicidal activity in neutrophils treated with testosterone at 10 mu M. Glutathione reductase activity was increased by testosterone treatment, whereas no effect was observed in other antioxidant enzyme activities. An increase in the content of thiol groups was observed at all testosterone concentrations. Lipid peroxidation in neutrophils evaluated by levels of TBARS was decreased at 10 nM and 0.1 mu M testosterone. These results indicate the antioxidant properties of testosterone in neutrophils as suggested by reduction of superoxide anion production, and lipid peroxidation, and by the increase in nitric oxide production, glutathione reductase activity and the content of thiol groups. Therefore, the plasma levels of testosterone are important regulators of neutrophil function and so of the inflammatory response. Copyright (C) 2010 John Wiley & Sons, Ltd.

Soybean and sunflower oil-induced insulin resistance correlates with impaired GLUT4 protein expression and translocation specifically in white adipose tissue

Free fatty acids are known for playing a crucial role in the development of insulin resistance. High fat intake is known for impairing insulin sensitivity; however, the effect of vegetable-oil injections have never been investigated. The present study investigated the effects of daily subcutaneous injections (100 mu L) of soybean (SB) and sunflower (SF) oils, during 7 days. Both treated groups developed insulin resistance as assessed by insulin tolerance test. The mechanism underlying the SB- and SF-induced insulin resistance was shown to involve GLUT4. In SB- and SF-treated animals, the GLUT4 protein expression was reduced similar to 20% and 10 min after an acute it? vivo stimulus with insulin, the plasma membrane GLUT4 content was similar to 60% lower in white adipose tissue (WAT). No effects were observed in skeletal muscle. Additionally, both oil treatments increased mainly the content of palmitic acid (similar to 150%) in WAT, which can contribute to explain the GLUT4 regulations. Altogether, the present study collects evidence that those oil treatments might generate insulin resistance by targeting GLUT4 expression and translocation specifically in WAT. These alterations are likely to be caused due to the specific local increase in saturated fatty acids that occurred as a consequence of oil daily injections. Copyright (C) 2010 John Wiley & Sons, Ltd.

Changes of glycogen content in liver, skeletal muscle, and heart from fasted rats

Glycogen content of white and red skeletal muscles, cardiac muscle, and liver was investigated in conditions where changes in plasma levels of non-esterified fatty acids (NEFA) occur. The experiments were performed in fed and 12 and 48 h-fasted rats. The animals were also submitted to swimming for 10 and 30 min. Glycogen content was also investigated in both pharmacologically induced low plasma NEFA levels fasted rats and pharmacologically induced high plasma NEFA levels fed rats. The participation of Akt and glycogen synthase kinase-3 (GSK-3) in the changes observed was investigated. Plasma levels of NEFA, glucose, and insulin were determined in all conditions. Fasting increased plasma NEFA levels and reduced glycogen content in the liver and skeletal muscles. However, an increase of glycogen content was observed in the heart under this condition. Akt and GSK-3 phosphorylation was reduced during fasting in the liver and skeletal muscles but it remained unchanged in the heart. Our results suggest that in conditions of increased plasma NEFA levels, changes in insulin-stimulated phosphorylation of Akt and GSK-3 and glycogen content vary differently in liver, skeletal muscles, and heart. Akt and GSK-3 phosphorylation and glycogen content are decreased in liver and skeletal Muscles, but in the heart it remain unchanged (Akt and GSK-3 phosphorylation) or increased (glycogen content) due to consistent increase of plasma NEFA levels. Copyright (C) 2009 John Wiley & Sons, Ltd.

Hepatic denervation impairs the assembly and secretion of VLDL-TAG

VLDL secretion is a regulated process that depends on the availability of lipids, apoB and MTP. Our aim was to investigate the effect of liver denervation upon the secretion of VLDL and the expression of proteins involved in this process. Denervation was achieved by applying a 85% phenol solution onto the portal tract, while control animals were treated with 9% NaCl. VLDL secretion was evaluated by the Tyloxapol method. The hepatic concentration of TAG and cholesterol, and the plasma concentration of TAG, cholesterol, VLDL-TAG, VLDL-cholesterol and HDL-cholesterol were measured, as well as mRNA expression of proteins involved in the process of VLDL assembly. Hepatic acinar distribution of MTP and apoB was evaluated by immunohistochemistry. Denervation increased plasma concentration of cholesterol (125.3 +/- 10.1 vs. 67.1 +/- 4.9 mg dL(-1)) and VLDL-cholesterol (61.6 +/- 5.6 vs. 29.4 +/- 3.3 mg dL(-1)), but HDL-cholesterol was unchanged (45.5 +/- 6.1 vs. 36.9 +/- 3.9 mg dL(-1)). Secretion of VLDL-TAG (47.5 +/- 23.8 vs. 148.5 +/- 27.4 mg dL h(-1)) and mRNA expression of CPT I and apoB were reduced (p < 0.01) in the denervated animals. MTP and apoB acinar distribution was not altered in the denervated animals, but the intensity of the reaction was reduced in relation to controls. Copyright (C) 2008 John Wiley & Sons, Ltd.

Quantification of chlordesmethyldiazepam by liquid chromatography-tandem mass spectrometry: application to a cloxazolam bioequivalence study

A rapid, sensitive and specific LC-MS/MS method was developed and validated for quantifying chlordesmethyldiazepam (CDDZ or delorazepam), the active metabolite of cloxazolam, in human plasma. In the analytical assay, bromazepam (internal standard) and CDDZ were extracted using a liquid-liquid extraction (diethyl-ether/hexane, 80/20, v/v) procedure. The LC-MS/MS method on a RP-C18 column had an overall run time of 5.0 min and was linear (1/x weighted) over the range 0.5-50 ng/mL (R > 0.999). The between-run precision was 8.0% (1.5 ng/mL), 7.6% (9 ng/mL), 7.4% (40 ng/mL), and 10.9% at the low limit of quantification-LLOQ (0.500 ng/mL). The between-run accuracies were 0.1, -1.5, -2.7 and 8.7% for the above mentioned concentrations, respectively. All current bioanalytical method validation requirements (FDA and ANVISA) were achieved and it was applied to the bioequivalence study (Cloxazolam-test, Eurofarma Lab. Ltda and Olcadil (R)-reference, Novartis Biociencias S/A). The relative bioavailability between both formulations was assessed by calculating individual test/reference ratios for Cmax, AUClast and AUCO-inf. The pharmacokinetic profiles indicated bioequivalence since all ratios were as proposed by FDA and ANVISA. Copyright (C) 2009 John Wiley & Sons, Ltd.

Alboserpin, a Factor Xa Inhibitor from the Mosquito Vector of Yellow Fever, Binds Heparin and Membrane Phospholipids and Exhibits Antithrombotic Activity

The molecular mechanism of factor Xa (FXa) inhibition by Alboserpin, the major salivary gland anticoagulant from the mosquito and yellow fever vector Aedes albopictus, has been characterized. cDNA of Alboserpin predicts a 45-kDa protein that belongs to the serpin family of protease inhibitors. Recombinant Alboserpin displays stoichiometric, competitive, reversible and tight binding to FXa (picomolar range). Binding is highly specific and is not detectable for FX, catalytic site-blocked FXa, thrombin, and 12 other enzymes. Alboserpin displays high affinity binding to heparin (K(D) similar to 20 nM), but no change in FXa inhibition was observed in the presence of the cofactor, implying that bridging mechanisms did not take place. Notably, Alboserpin was also found to interact with phosphatidylcholine and phosphatidylethanolamine but not with phosphatidylserine. Further, annexin V (in the absence of Ca(2+)) or heparin outcompetes Alboserpin for binding to phospholipid vesicles, suggesting a common binding site. Consistent with its activity, Alboserpin blocks prothrombinase activity and increases both prothrombin time and activated partial thromboplastin time in vitro or ex vivo. Furthermore, Alboserpin prevents thrombus formation provoked by ferric chloride injury of the carotid artery and increases bleeding in a dose-dependent manner. Alboserpin emerges as an atypical serpin that targets FXa and displays unique phospholipid specificity. It conceivably uses heparin and phosphatidylcholine/phosphatidylethanolamine as anchors to increase protein localization and effective concentration at sites of injury, cell activation, or inflammation.

Effect of endurance training upon lipid metabolism in the liver of cachectic tumour-bearing rats

The syndrome of cancer cachexia is accompanied by several alterations in lipid metabolism, and the liver is markedly affected. Previous Studies showed that moderate exercise training may prevent liver fill accumulation through diminished delivery of lipids to the liver, increased hepatic oxidation and increased incorporation of triacylglycerol (TAG) into very low density lipoprotein (VLDL). Our aim was to examine the influence of moderate intensity training (8 weeks) upon TAG content, VLDL assembly and secretion, apolipoprotein B (apoB) and microsomal transfer protein (MTP) gene expression in the liver of cachectic tumour-bearing rats. Animals were randomly assigned to a sedentary control (SC), sedentary tumour-bearing (ST) or exercise-trained control (EC) or to all exercise trained tumour-bearing (ET) group. Trained rats ran on a treadmill (60% VO2max) for 60 min day(-1), 5 day week(-1), for 8 weeks. TAG content and the rate of VLDL secretion (followed for 3 h), its well its mRNA expression of apoB and MTP, and total cholesterol, VLDL-TAG, VLDL-cholesterol, high density lipoprotein cholesterol (HDL-cholesterol) and tumor weight were evaluated. VLDL-cholesterol showed a decrease in ST (p < 0.05) in relation to SC. Serum TAG, VLDL-TAG and tissue TAG content were all increased in ST (p < 0.01), when compared with SC. ST showed a lower rate of VLDL secretion (p < 0.05) and reduced expression of apoB (p < 0.001) and MTP (p < 0.001), when compared with SC. These parameters were restored to control values (p < 0.05) when the animals were submitted to the exercise training protocol. Tumour weight decreased 10-fold after training (p < 0.001). It is possible to affirm, therefore, that endurance training promoted the re-establishment of lipid metabolism in cachectic tumour-bearing animals, especially in relation to VLDL secretion and assembly. Copyright (C) 2008 John Wiley & Sons, Ltd.

The importance of hormone receptor analysis in osteosarcoma cells growth submitted to treatment with estrogen in association with thyroid hormone

Bone tumor incidence in women peaks at age 50-60, coinciding with the menopause. That estrogen (E2) and triiodothyronine (T3) interact in bone metabolism has been well established. However, few data on the action of these hormones are available. Our purpose was to determine the role of E2 and T3 in the expression of bone activity markers, namely alkaline phosphatase (AP) and receptor activator of nuclear factor kappa B ligand (RANKL). Two osteosarcoma cell lines: MG-63 (which has both estrogen (ER) and thyroid hormone (TR) receptors) and SaOs-29 (ER receptors only) were treated with infraphysiological E2 associated with T3 at infraphysiological, physiological, and supraphysiological concentrations. Real-time RT-PCR was used for expression analysis. Our results show that, in MG-63 cells, infraphysiological E2 associated with supraphysiological T3 increases AP expression and decreases RANKL expression, while infraphysiological E2 associated with either physiological or supraphysiological T3 decreases both AP and RANKL expression. On the other hand, in SaOs-2 cells, the same hormone combinations had no significant effect on the markers` expression. Thus, the analysis of hormone receptors was shown to be crucial for the assessment of tumor potential growth in the face of hormonal changes. Special care should be provided to patients with T3 and E2 hormone receptors that may increase tumor growth. Copyright (C) 2007 John Wiley & Sons, Ltd.

MicroRNA expression is differentially altered by xenobiotic drugs in different human cell lines

Several noncoding microRNAs (miR or miRNA) have been shown to regulate the expression of drug-metabolizing enzymes and transporters. Xenobiotic drug-induced changes in enzyme and transporter expression may be associated with the alteration of miRNA expression. Therefore, this study investigated the impact of 19 xenobiotic drugs (e. g. dexamethasone, vinblastine, bilobalide and cocaine) on the expression of ten miRNAs (miR-18a, -27a, -27b, -124a, -148a, -324-3p, -328, -451, -519c and -1291) in MCF-7, Caco-2, SH-SY5Y and BE(2)-M17 cell systems. The data revealed that miRNAs were differentially expressed in human cell lines and the change in miRNA expression was dependent on the drug, as well as the type of cells investigated. Notably, treatment with bilobalide led to a 10-fold increase of miR-27a and a 2-fold decrease of miR-148a in Caco-2 cells, but no change of miR-27a and a 2-fold increase of miR-148a in MCF-7 cells. Neuronal miR-124a was generally down-regulated by psychoactive drugs (e. g. cocaine, methadone and fluoxetine) in BE(2)-M17 and SH-SY5Y cells. Dexamethasone and vinblastine, inducers of drug-metabolizing enzymes and transporters, suppressed the expression of miR-27b, -148a and -451 that down-regulate the enzymes and transporters. These findings should provide increased understanding of the altered gene expression underlying drug disposition, multidrug resistance, drug-drug interactions and neuroplasticity. Copyright (C) 2011 John Wiley & Sons, Ltd.

The novel ruthenium-gamma-linolenic complex [Ru(2)(aGLA)(4)Cl] inhibits C6 rat glioma cell proliferation and induces changes in mitochondrial membrane potential, increased reactive oxygen species generation and apoptosis in vitro

The present study reports the synthesis of a novel compound with the formula [Ru(2)(aGLA)(4)Cl] according to elemental analyses data, referred to as Ru(2)GLA. The electronic spectra of Ru(2)GLA is typical of a mixed valent diruthenium(II,III) carboxylate. Ru(2)GLA was synthesized with the aim of combining and possibly improving the anti-tumour properties of the two active components ruthenium and gamma-linolenic acid (GLA). The properties of Ru(2)GLA were tested in C6 rat glioma cells by analysing cell number, viability, lipid droplet formation, apoptosis, cell cycle distribution, mitochondrial membrane potential and reactive oxygen species. Ru(2)GLA inhibited cell proliferation in a time and concentration dependent manner. Nile Red staining suggested that Ru(2)GLA enters the cells and ICP-AES elemental analysis found all increase in ruthenium from <0.02 to 425 mg/Kg in treated cells. The sub-G1 apoptotic cell population was increased by Ru(2)GLA (22 +/- 5.2%) when analysed by FACS and this was confirmed by Hoechst staining of nuclei. Mitochondrial membrane potential was decreased in the presence of Ru(2)GLA (44 +/- 2.3%). In contrast, the cells which maintained a high mitochondrial membrane potential had an increase (18 +/- 1.5%) in reactive oxygen species generation. Both decreased mitochondrial membrane potential and increased reactive oxygen species generation may be involved in triggering apoptosis in Ru(2)GLA exposed cells. The EC(50) for Ru(2)GLA decreased with increasing time of exposure from 285 mu M at 24h, 211 mu M at 48 h to 81 mu M at 72 h. In conclusion, Ru(2)GLA is a novel drug with anti proliferative properties in C6 glioma cells and is a potential candidate for novel therapies in gliomas. Copyright (C) 2009 John Wiley & Sons, Ltd.